Diffusible guidance molecules are responsible for guiding growing axons towards their target cells in the developing nervous system. Several families of guidance factors and their receptors have been identified and the signaling mechanisms are beginning to be elucidated. In this project, we propose to continue our in vitro study of signal transduction events underlying the turning responses of the axonal growth cones of cultured Xenopus spinal neurons and rat cerebellar granule cells induced by microscopic gradients of two guidance molecules, netrin-1 and brain-derived neurotrophic factor (BDNF), and by a secreted form of myelin-associated glycoprotein (MAG). In part I, we will continue our study of the spatiotemporal profiles of Ca2+ and cAMP signals at the growth cone triggered by guidance cues, and the causal relationship betweenCa2+ and cAMP signals that lead to attractive and repulsive turning of the growth cone. In Part II, we will examine how Ca2+ elevation triggered by the activation of the membrane receptors of guidance factors is linked to the activation of the Rho family of small guanosine triphosphatases (GTPases), which are known to regulate the cytoskeletal rearrangements underlying growth cone turning. In Part III, we will examine the initial cytoplasmic events triggered by a gradient of guidance factors that are likely to serve for the amplification of guidance signals, with particular focus on phosphoinositol 3 kinase and its effector molecules that may undergo rapid accumulation towards the leading edge of the growth cone. The role of Ca2+ signaling and Rho GTPases in the amplification process will also be addressed. In Part IV, we will further examine the phenomenon of adaptation (desensitization and resensitization) of growth cone responses in the presence of a gradient of the guidance factor, and the cellular mechanisms underlying the desensitization and resensitization processes. In particular, we will examine how local translation of specific proteins at the growth cone participates in the adaptation process. Together, these studies offer a unique opportunity for elucidating the cellular transduction events at the growth cone underlying the action of extracellular diffusible guidance molecules. The results will contribute to our understanding of the development of the nervous system and provide insights into potential therapeutic approaches for promoting nerve regeneration after injury.